Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters

Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, are a necessary basis for fluorescence-based super-resolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans–cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time resolution and is thus broadly applicable to different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional useful strategy for fluorescence-based multiplexing that can be implemented on a broad range of both stationary and moving samples.

extracted with a 3:1 choloroform/methanol solution.The dye-labeled lipids were then used to prepare small unilamellar vesicles (SUVs), as previously described [1].In short, POPC in chloroform (1.74 mol, Avanti Polar Lipids, Inc.) was dried under a gentle flow of N 2 for about μ 20 min.After complete removal of any residual solvent, 1.4 ml DPBS buffer was added to the dried dye lipids, vortexed for 1 min, and then sonicated at 0 o C for 10 min by a Branson SFX250 sonicator at 50% duty cycle (0.50 s on/off), 50% power (125 W) and a 1/8" microtip (Emerson Electric Co, St. Louis, MO, USA).After sonication, the solution was centrifuged for 20 min at 14000 g, and the supernatant was then filtered by using a 0.2 m spin-filter (Corning, NY, USA) μ to remove large aggregates.The fraction of labelled POPE lipids in the SUVs were typically lower than 1/50000, to prevent having more than one fluorophore per SUV.
For the immunostaining, the cell culture medium was first removed and HEK293 cells were washed 3 times with DPBS.The cells were fixed and permeabilized with 3.7% paraformaldehyde (PFA), 0.1% glutaraldehyde and 0.5% Triton X for 20 min, and then washed 3 times with DPBS.Afterwards, the cells were blocked for nonspecific binding with 2% w/v bovine serum albumin (Sigma, Sweden) for about 40 min and washed three times with DPBS.
The cells were incubated with primary antibodies (1:200) for 1 h.Rabbit polyclonal antibodies (Invitrogen) were used for targeting nucleoporin Nup153 while Mouse monoclonal antibodies (Invitrogen) were used against alpha tubulin of HEK293 cells.The cells were washed three times with DPBS before incubating with labelled secondary antibodies for an hour (goat anti-rabbit and goat anti-mouse (Sigma), conjugated with Abberior Star 635 and Cy5 fluorophores, respectively).The labelled cells were washed 3 times and DPBS was used as an observation medium for experiments.

Section S3. Experimental setup for stationary wide-field TRAST measurements, data acquisition and analysis.
TRAST measurements were carried out on a home-built TRAST setup, as previously described [1,2], based on an inverted epi-fluorescence microscope (Olympus, IX70).Fluorescence was excited by a 638 nm diode laser (Cobolt, 06-MLD, 240 mW) using an excitation filter (Semrock BrightLine 637/7).The laser beam was modulated by an acousto-optic modulator (AOM; AA Opto Electronics, MQ180-A0,25-VIS), reflected by a dichroic mirror (ZT640rdc) and then focused close to the back aperture of the objective (Olympus, UPLSAPO 60x/1.20 W) to produce a wide-field illumination in the sample (beam waist ω 0 = 10-25 µm (1/e 2 radius).The fluorescence signal was collected by the same objective, passed through the same dichroic mirror and an emission filter (ET706/95m, Chroma) before detection by a sCMOS camera (Hamamatsu ORCA-Flash4.0V3).The experiments were controlled and synchronized by custom software implemented in Matlab.A digital I/O card (PCI-6602, National Instruments) was used to trigger the camera and generate random excitation pulse trains sent to the AOM driver unit.
In the data acquisition, a complete TRAST experiment consisted of a stack of 30 fluorescence images.Each image represents the total fluorescence signal from an entire excitation pulse train, captured using a camera exposure time of .Pulse durations, , were distributed   =     logarithmically between 100 ns and 10 ms and were measured in a randomized order to avoid bias due to time effects.An additional 10 reference frames, all using 100 ns pulse duration (w short ) to avoid dark state build-up, were inserted at regular intervals between the 30 main images to track any permanent bleaching of the sample.
The TRAST data was analyzed by software implemented in Matlab, as previously described [2,3].Recorded TRAST data was first pre-processed by subtraction of static ambient background, by optional binning to either larger pixels or regions of interest (ROIs) within the recorded images, and by correction for bleaching.The bleaching correction was based on 10 reference frames, recorded in between the regular frames throughout the measurements.The overall bleaching was maximally 5-10 % of the total detected intensity in the experiments.
In all measurements, TRAST curves were produced by calculating within a region 〈  ()〉  of interest (ROI) corresponding to a radius in the sample plane, centered on the 15 μm excitation beam, for both the vesicle and live cell measurements.Within the selected ROI, an average excitation rate was then calculated, as described in section S4.Fitting of photophysical parameters was then performed by simulating theoretical TRAST curves using Eqs.(1-3) and comparing them to the experimental data.The set of parameters best describing the experimental data was then found using non-linear least squares optimization.
The non-uniform shape of the excitation beam means that the excitation photon flux, , is Φ  () a function of position in the sample.As a consequence, a detailed TRAST analysis should include a spatial dependence to both the excitation rates and the resulting electronic state populations.The total fluorescence signal on each pixel of the camera then becomes a convolution of and the microscope collection efficiency function, , as shown in Eq. S 1 (t) () Above equation, represents the population of at onset of  1 () =  01 ()/( 01 () +  10 ) S excitation, after equilibration between the singlet states and , but before build-up of the S 0 S 1 other states.

Section S5. Determination of flow profiling by microfluidic FCS measurements
To determine the flow profiling in flow-based TRAST measurements, complementary FCS experiments were performed with a microfluidic system.By scanning the position of the excitation laser with 5 m spatial resolution across the flow direction in the middle height (that is μ 25 m) of the flow channel, 98 different FCS curves were recorded for 10 nM of CF640R dye in μ PBS under a constant flow rate (40 L/min) by using a mechanical syringe pump.In microfluidic μ FCS measurements, excitation power is kept low at 84 W to avoid the triplet state build-up.μ Some example FCS data is visualized in Figure S2A where all experimental FCS curves were fitted to a modified correlation function as given below [4]:  , can be described by: () where denotes the translational diffusion-dependent part signifies the contribution   () () from photo-induced dark state transitions.can be expressed as: with and denoting the distances from the center of the laser beam focus in the radial and     axial direction respectively at which the collected fluorescence intensity has dropped by a factor of compared to its peak value.is the mean number of fluorescent molecules within the       detection volume.is the characteristic diffusion time of the fluorescent molecules, given by   the diffusion coefficient as .   = If no dark state transitions occur, the blinking term .Otherwise, for a fluorophore with   () = 1 n dark transient states, and for much longer than the anti-bunching relaxation times of the  fluorophores, can be expressed as a normalized set of relaxation terms [5,6], averaged   () over the confocal detection volume, weighted by the square of the detected molecular brightness of the molecules, : Here, are the eigenvalues and the related amplitudes, reflecting the population build-  () () up of the different photo-induced non-fluorescent states.At steady state and with no photobleaching, the sum of the population probabilities for S 0 and S 1 , together with equals one.
For FCS-analysis of single Cy5-and CF640R-labelled SUVs alone and in different mixtures, the Sympohotime software (Picoquant, Berlin) and an upper threshold was applied to filter out bursts/spikes in the detected fluorescence intensity time-traces before calculating the FCS curves.The threshold was set to 7 standard deviations above the mean fluorescence intensity.
Aggregates or multi-labelled SUVs passing through the FCS detection volume, can result in lower dark state relaxation amplitudes than from the single-labelled, not aggregated SUVs.Since they would also contribute with their brightness squared to the experimental FCS curves (Eq.13), the use of filtered FCS curves allowed this contribution to be minimized and made the estimation of the relative concentrations of Cy5 and CF640R more reliable.
For FCS-analysis of single Cy5-and CF640R-labelled SUVs alone and in different mixtures, the Sympohotime software (Picoquant, Berlin) and an upper threshold was applied to filter out bursts/spikes in the detected fluorescence intensity time-traces before calculating the FCS curves.The threshold was set to 7 standard deviations above the mean fluorescence intensity.
Aggregates or multi-labelled SUVs passing through the FCS detection volume, can result in lower dark state relaxation amplitudes than from the single-labelled, not aggregated SUVs.Since they would also contribute with their brightness squared to the experimental FCS curves (Eq.13), the use of filtered FCS curves allowed this contribution to be minimized and made the estimation of the relative concentrations of Cy5 and CF640R more reliable.While recording TRAST-curves of Cy5, it was noted that the amplitudes of the curves were generally reduced compared to the dark state relaxation amplitudes obtained in the FCS measurements (Figure 2F versus Figure 2H).One possible reason for this is incomplete dark state relaxation in-between the excitation pulses applied in the TRAST measurements.In the stationary wide-field TRAST experiments, the duty-cycle, η, was set to 0.01.For most fluorophores, this gives sufficient time for their dark transient states to fully recover back to the emissive singlet state.At our experimental conditions, it was found that for Cy5, the backisomerization rate is predominantly excitation-driven (Eq.10), with only a lower thermal recovery rate, , effective in-between the pulses.To investigate to what extent η affects the  ℎ  dark state relaxation amplitudes in the recorded TRAST curves and may lead to incomplete recovery from P to N in-between the excitation pulses, TRAST-curves were recorded with different η.At our experimental conditions, it was found that η at around 0.001 was required for the TRAST-curves to converge to similar data points (Figure S1A).An incomplete recovery back to the ground state through thermal relaxation can likely explain the reduction in amplitude observed for higher η. Figure S1B shows how the normalized average fluorescence varies with η for different pulse-widths, w, confirming the convergence of data-points for η <0.001 in Figure S1A.One can also note that η primarily influences the shorter w data points, with the time inbetween pulses then shorter and giving less time for ground state recovery.In Figure S1C, the  1 population of Cy5 is calculated based on the fitted isomerization rates, as obtained by FCS, with a thermal rate of µs -1 and with the same duty-cycles as was used for the  ℎ = 0.005 measurements in Figure S3A.The calculated curves in Figure S1C 6) where is the flow time of the CF640R dyes in microfluidic channel.In curve fitting, N and   were fitted as free parameters whereas is fixed to 50 s (as determined from the FCS     μ curve recorded at 40 L/min flow rate).With the knowledge of and values, the flow μ  0   speed at different coordinates is calculated using .The fitted N and     =  0 /    values together with calculated were plotted in Figure S2(B-D).For FCS curves recorded   at the coordinates of -240 m and 240 m, smaller FCS amplitudes were found most likely due μ μto the sticking of dye molecules to the interior walls of microfluidic chip (the flow channel width is 500 m).Therefore, N values were found to be higher for the coordinates closer to the channel μ walls (see FigureS2B).However, in the middle region of the flow channel between -180 m and μ 180 m, the fitted and calculated values were found to be fairly flat, demonstrating a μ     uniform laminar flow profile.In flow-based TRAST experiments, as the excitation beam curtains are well positioned in between these coordinates, the flow profile is treated as laminar flow.

Figure S1 .
Figure S1.FCS measurements performed with microfluidic flow system.(A) Experimental FCS curves recorded at different coordinates across the flow direction.(B) Number of molecules in the detection volume, (C) flow time, and (D) flow speed, of CF640 molecules in     microfluidic channel as obtained from the fitting of FCS curves at different channel coordinates.
of Cy5 from its photo-isomerized state P -influence of excitation duty cycle.
Figure S2.(A) TRAST curves recorded from Cy5 in solution with different η applied (indicated in the legend).Here, 4.6 kW/cm².(B) TRAST measurements of Cy5 in solution with Φ exc = different fixed pulse-widths (indicated in legend), versus η ( 4.6 kW/cm²).(C) Calculated Φ exc = TRAST-curves for different η, based on fitted isomerization rate parameter values determined from FCS, and with µs -1 .(D) Initial populations of for different η versus w.  ℎ  = 0.005  0 . However, simulating the whole 3D sample volume, and computing the projected 2D image on the camera, becomes a costly operation when performed in each iteration of the fitting algorithm.While this procedure is possible, and sometimes required, pre-computing an average